The controls governing S-nitrosation are unknown. In addition, the ramifications of S-nitrosation signaling are virtually unknown. Determining the molecular mechanism(s) that permits cells to achieve specificity in S-nitrosation reactions is the focus of this proposal. Nitric oxide (NO) plays integral roles in mammalian physiology including vasodilation, neuronal signaling, and immunity. NO affects cellular physiology by multiple pathways. The best studied pathway is through binding to the enzyme, soluble guanylate cyclase (sGC). The actions of NO that have been described cannot be completely accounted for when only considering sGC as a target. S-Nitrosation is one type of sGC-independent signaling and involves the post-translational modification of cysteine on proteins. In many cases, modification of a cysteine alters protein function. Processes similar to this are almost exclusively a regulated cellular event with a biological machinery in tight control. In vitro work has shown that when NO reacts with a protein, many cysteine thiols are modified. However, in a cellular context when NO was not added but produced by the cell itself, multiple modifications never occur. Additionally, NO is synthesized at very low concentrations such that without a control mechanism in place, protein modification would be highly inefficient. The most logical explanation for such disparities is that the in vitro experiment lacked the cellular components that confer specificity to the S-nitrosation reaction. Experimentally, this project will attempt to identify these components by using a variety of advanced tools such as: tailored affinity probes, inductively-coupled plasma spectroscopy, fluorescence spectroscopy, and recently developed S-nitrosation specific biochemical assays. Nitric oxide (NO) mediates blood vessel relaxation, complex aspects of myocardial function, perfusion and function of all major organs, synaptic plasticity in the brain, platelet aggregation, skin function, and numerous other physiological processes. Given the role of NO in human biology, a complete understanding of the molecular details involved in its signaling will have clear application to the understanding and treatment of a broad spectrum of diseases, such as hypertension and cardiovascular disease. This research can lead to the development of more effective therapies and, potentially, reduce health care costs.